- Email: [email protected]
Effects of soy proteins and hydrolysates on fat globule coalescence and meltdown properties of ice cream
Accepted Manuscript Effects of soy proteins and hydrolysates on fat globule coalescence and meltdown properties of ice cream Wenpu Chen, Guijiang Liang, Xiang Li, Zhiyong He, Maomao Zen, Daming Gao, Fang Qin, H. Douglas Goff, Jie Chen PII:
S0268-005X(18)32290-2
DOI:
https://doi.org/10.1016/j.foodhyd.2019.02.045
Reference:
FOOHYD 4971
To appear in:
Food Hydrocolloids
Received Date: 22 November 2018 Revised Date:
19 February 2019
Accepted Date: 25 February 2019
Please cite this article as: Chen, W., Liang, G., Li, X., He, Z., Zen, M., Gao, D., Qin, F., Goff, H.D., Chen, J., Effects of soy proteins and hydrolysates on fat globule coalescence and meltdown properties of ice cream, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.02.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Protein
Soy
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Native
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Graphical abstract
Isolate
pepsin (SPHPe)
protein
hydrolyzed
by
papain (SPHPa)
Commercial Soy Protin (CSPI)
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Isolate
Composition and properties of soy protein isolate and its hydrolysate
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hydrolyzed
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The interaction between soy protein subunits and monoglycerides at oil-water interface
protein
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(NSPI)
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Wenpu Chenab , Guijiang Liangab, XiangLic, Zhiyong Heab ,Maomao Zenab ,Daming Gaoab, Fang
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Qin ab, H. Douglas Goffd, Jie Chen*ab
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a
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China
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b
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, PR, China
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School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR, China
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Department of Food Science, University of Guelph, ON N1G2W1, Canada
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Effects of soy proteins and hydrolysates on fat globule coalescence and meltdown properties of ice cream
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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR.
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*Corresponding author:
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Professor Jie Chen
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State Key Laboratory of Food Science and Technology, School of Food Science and Technology,
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Jiangnan University, Wuxi, Jiangsu, 214122, China
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Tel.: +86-510-85329032
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Fax: +86-510-85919065
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E-mail address: [email protected]
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Abstract
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The effect of soy protein isolate and its hydrolysates on ice cream mix stability and melt-down
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properties of ice cream were investigated. Ice creams were made with 10% milk fat, 3.5%
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protein and 34.3% total solids, and all contained 0.15% added monoglycerides. The proteins
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used were native soy protein isolate (NSPI), commercial soy proteins isolate (CSPI), soy protein
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hydrolyzed by pepsin (SPHPe), soy protein hydrolyzed by papain (SPHPa) and skim milk
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powder (SMP). Ice cream with SPHPe containing the highest relative composition of β-subunit
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showed good mix emulsion stability and rapid melting rate because β-subunit cannot be
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displaced by the monoglycerides, leading to lack of fat partial coalescence in the ice cream.
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SPHPa ice cream exhibited comparable functionality to SMP in rheological and meltdown
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properties. SDS-PAGE results indicated that α subunit, α' subunit, acidic subunit, basic subunit
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and small molecule polypeptide were displaced by monoglycerides during ice cream aging.
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Selective hydrolysis of soy protein can be used for ice cream with sufficient fat partial
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coalescence and good melt-down rates.
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Key Word： Soy Protein Isolate, Hydrolysates, Ice Cream
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Introduction
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Ice cream is a complex food emulsion system with ice crystals, dispersed fat globules and air
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cells, protein-hydrocolloid structures and a continuous unfrozen aqueous phase. A higher degree
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of fat destabilization (a high level of partially coalesced fat globule clusters) stabilizes the air
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phase and contributes to the physical attributes of ice cream such as texture and melting
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properties (Goff & Hartel, 2013). Traditionally, milk protein is a major source in making ice
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cream due to its multiple desirable functionalities including stabilizing fat droplets by steric
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repulsion, interaction with emulsifiers at the interface, stabilizing air bubbles and enhancing
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unfrozen phase viscosity to prevent the growth of larger ice crystals besides its nutritional
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function (Dickinson, 2003; Goff & Hartel, 2013). The role of milk protein in ice cream has been
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fully explored in shape retention, fat globule destabilization and melting properties (Alvarez,
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Wolters, Vodovotz, & Ji, 2005; Daw & Hartel, 2015; Patel, Baer, & Acharya, 2006).
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Soy protein is considered as an alternative to or extension of milk protein in food systems due to
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its availability, emulsifying properties, plant-derived source and health benefits (Abdullah et al.,
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2003; Anderson, Smith, & Washnock, 1999; Biswas, Chakraborty, & Choudhuri, 2002). As a
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macromolecular emulsifier with amphiphilic nature, soy protein isolate stabilizes oil-water or air-
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water interfaces by forming a viscoelastic film at the oil/water interface (Palazolo, Mitidieri, &
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Wagner, 2003; Puppo et al., 2008). The characteristics of soy protein isolate such as solubility
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and emulsification make it possible for it to be used in ice cream (Dervisoglu, Yazici, &
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Aydemir, 2005; Friedeck, Aragul-Yuceer, & Drake, 2003). The addition of soy protein isolate in
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ice cream reduces the effect of heat shock on ice recrystallization and melting rate (Pereira et al.,
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2011). Replacing partial skim milk powder with soy extract in ice cream achieved smaller ice
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crystals and minimized ice recrystallization under temperature fluctuations (Pereira et al., 2011).
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However, 100% soy protein isolate as the protein source in the formula led to high viscous mix
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and meltdown-resistant ice cream (Akesowan, 2009; Dervisoglu et al., 2005). There has been
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some published research about soy protein functionality in the formation of ice cream structure.
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Soy protein with larger molecular weight and compact structure had limited surface activity at
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the oil/water interface due to slow rate of diffusion and adsorption (Santiago et al., 2008), which
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may impact the formation of partial fat coalescence during ice cream freezing. The production of
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high-quality ice cream with soy protein would therefore require comparable ice cream mix
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viscosity as milk protein and the formation of partial fat coalescence, which are necessary for a
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stable foam structure in ice cream, shape retention dryness and slowness of meltdown (Goff,
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1997). Optimization of functionalities of soy protein structure is of interest to achieve a high
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degree of fat globule partial coalescence in ice cream making.
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Limited enzymatic hydrolysis of soy protein has been used to expose concealed hydrophobic
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groups and increase molecular flexbility, which indicates good affinity at the oil/water interface
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(Conde & Patino, 2007; Panyam & Kilara, 1996). Huang, Catignani, & Swaisgood (1996)
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reported that limited enzymatic hydrolysis with pepsin increased emulsifying activity index and
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achieved smaller fat globules compared to intact soy protein. The objective of this study was to
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apply pepsin and papain to modify soy protein for hydrolysates with different viscoelastic and
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interfacial properties and identify the main soy protein composition that may impact ice cream
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microstructure and meltdown properties. For this purpose, ice cream and its mixes formulated
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with different soy protein hydrolysates were characterized by rheology, particles size distribution,
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the amount and peptide profile of adsorbed protein at oil/water interface, meltdown rate and
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structure by confocal microscopy.
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2.0. Materials and methods
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2.1. Materials
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Soy bean was obtained from local market (Wuxi, China). Soy protein isolate (SPI) was prepared
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according to the procedure given by Diftis & Kiosseoglou (2006). Kjeldahl method (N x 6.25)
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was used to determine protein content of the samples (Ryan et al., 2008). Commercial soy
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protein isolate (CSPI) were obtained from Solae, USA. Skim milk powder (SMP), unsalted
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butter (Anchor) and lactose were obtained from Fonterra, New Zealand. The main composition
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of SMP includes 35% milk protein, minerals, lactose, and less than 1% fat content. Corn syrup
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solids (36DE) was obtained from Cargill, China. Guar gum, locust beam guar and
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monoglycerides were obtained from Danisco, China. Pepsin (3000 U/mg) was purchased from
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Sangon Biotech, Shanghai, China and papain (2000 U/mg) was purchased from Regal, Shanghai,
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China. Deionized water was used as the ingredient water and all other chemicals used were
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analytical grade unless otherwise specified.
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2.2. Preparation and characterization of soy protein isolate and its hydrolysates
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Commercial soy protein isolate (CSPI) was reconstituted and centrifuged to remove the insoluble
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portion (320 x g, 5 min, 25°C). The supernatant was freeze-dried for powder. For native soy
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protein isolate and its hydrolysates, defatted soy protein flakes were dispersed in a 9-fold weight
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of distilled water at 25°C, and pH value of the slurry was adjusted to 8.0 with 2M NaOH. The
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slurry was stirred for 120 min and centrifuged (10000 x g, 20 min, 25°C) to remove the insoluble
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portion. The supernatant was adjusted to pH 4.5 with 2M HCl and acid-precipitated soy protein
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curd was collected by centrifugation (3300 x g, 10 mins, 25°C). Three portions of acid-
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precipitated soy protein curd were prepared. The first portion was made as native soy protein
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isolate (NSPI). The precipitate was dispersed in a 4-fold weight of water and neutralized to pH
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7.0 with 2M NaOH before freeze drying.
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The second portion of curd was dispersed in water to the final concentration 5% (w/v) at 40°C
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and pH was adjusted to 2.0 with 2M HCl. Pepsin was added to the SPI dispersion with enzyme-
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to-SPI ratio of 0.3 wt% with stirring. The slurry was incubated at 40°C for 2h and was
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terminated by the heat treatment (120 °C, 15s). The collected precipitate was dispersed in a 4-
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fold weight of distilled water and neutralized to pH 7.0 with 2M NaOH before freeze drying. It
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will be referred to hereafter as soy proteins hydrolyzed by pepsin (SPHPe). The third portion of
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curd was dispersed in water to the concentration 5% (w/v) and adjusted to pH 7.0 with NaOH.
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0.5 wt% papain was added and incubated for 30 min at 50°C. The reaction was terminated by
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heat treatment (120°C, 15s). The solution was freeze-dried to obtain soy proteins hydrolyzed by
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papain (SPHPa). The protein content in NSPI, CSPI, SPHPe and SPHPa are 79.5%, 81.4%, 68.0%
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and 77.8%. The rest of the main composition in NSPI, CSPI, SPHPe and SPHPa are soluble
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carbohydrate, minerals and very small amount of soy oil residue.
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The degree of hydrolysis (DH) was determined by TNBS method described by Adler-Nissen
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(1979). Emulsifying activity index (EAI) and emulsion stability index (ESI) of the samples were
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determined by the method described by Guo et al. (2015).
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The polypeptide profiles of soy protein isolate and its hydrolysates were determined by sodium
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dodecyl sulphate-polyarylamide gel electrophoresis (SDS-PAGE) according to the procedure
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given by Li et al. (2016) and Keerati-u-rai & Corredig (2009), with slight modification. A mini-
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protein electrophoresis system (Bio-Rad Laboratories, Hercules, CA, U.S.A.) was used for
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analysis. For the raw ingredients, soy protein isolate and its hydrolysates were dissolved in the
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buffer (0.0625M of Tris-HCl, 10% glycerin, 2% SDS and 0.0025% bromophenol blue) to 1 mg/
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mL protein solution. After heating for 3 min in boiling water and centrifuging at 5000 x g for 10
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min at room temperature with a TGL-16G 144 centrifuge (Anting Scientific Instrument Co. Ltd.,
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Shanghai, China), aliquots (20 µL) of the prepared samples were loaded onto the gels. For
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adsorbed protein on the fat globules in the thawed ice creams, the mixes were centrifuged at
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10000g for 45 min at 25 °C in a temperature-controlled centrifuge (SIGMA 3K15, SIXMA,
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Germany). Cream was collected and was dried on the filter paper. Collected cream was re-
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suspended with ultrapure water to the initial volume then mixed with equal amount of buffer for
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composition determination using electrophoresis. Coomassie Brilliant Blue (G-250) was used to
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stain the gel. All gels were scanned by a computing densitometer (Molecular Imager
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ChemiDocXRS+, Bio-Rad, USA) Image Lab software (Bio-Rad, USA) was used to integrate the
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intensities of bands.
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2.3. Formulation of ice cream and processing
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Ice cream mixes were prepared with the formulation in Table 1 with the same protein content
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(3.5% w/w). Each ice cream mix was pasteurized at 85 °C for 1 min. The mix subsequently went
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through 2- stage homogenizer (17.2/3.4 MPa) (NANO Homogenize Machine, Model AH-Basic,
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ATS Engineering Inc). The mix was immediately cooled to 4°C and aged for 24h at 4°C. Ice
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cream was produced in batch freezer (Model 104, Taylor Freezers, Rockton, IL, USA) with
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outlet temperature between - 5 and - 6 °C. The overrun of ice cream was in the range of 40-50%.
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Ice creams were collected in paper containers with weight around 90-100 g and hardened below
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-30 °C in freezer for at least 72h before further testing.
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2.4. Rheological properties
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The rheological profile of all ice cream mixes were measured in a HAAKE MARS Rheometer
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(Thermo Scientific, Germany) equipped with cone and plate (40 mm, 2°). Sample size was 2 mL.
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Samples were presheared at 20 s-1 for 30s and the shear stress was recorded at shear rate from 0s-1
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to 300s-1 over 420s then a decreasing shear rate to 0s-1 over 420s. Rheological data were
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modelled according to Herschel-Bulkley model. τ =τ0 + K • n
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where τ is shear stress, K is consistency coefficient, γ∙ is shear rate, n is flow behavior index and
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τ0 is the yield stress.
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2.5. Particle size distribution
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Particle size distribution of fresh and melted ice cream mixes were determined by Microtrac
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BlueWave S3500 Laser Diffraction Particle Size Analyzer (Microtrac Inc. Montgomeryville, PA,
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USA). Samples were directly diluted (1:1000) in the sample chamber with MiliQ water at 16-
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18°C. The refractive index of the emulsion was taken as 1.33. Measurements were performed at
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ambient temperature and repeated in triplicate. Volume weighted mean (d4,3) were recorded.
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2.6. Adsorbed protein fraction determination
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The fraction of adsorbed protein (AP %) of fresh and thawed samples were determined using the
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method described by Ye (2008) and Chen et al. (2019) with slight modification. Each sample
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(4mL) was centrifuged at 10000g for 45 min at 25 °C in a temperature-controlled centrifuge
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(SIGMA 3K15, SIXMA, Germany). After the centrifugation, the aqueous layer was carefully
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removed using a syringe. The cream layer was re-dispersed in deionized water and centrifuged at
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10000g for 45 min at 25 °C. The subnatants were filtered through 0.22 um filters (Milipore
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Corp.). Kjeldahl method (N x 6.25) (KDN-103F, Automatic Nitrogen Determinator, Qianjian,
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Shanghai, China) was used to determine the filtrates and the total protein in the mix. Adsorbed
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protein fraction (%) was calculated by the formula below:
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Adsorbed protein fraction (%) = (C0- Ci)/C0 x 100%
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where C0 is the initial protein concentration of the emulsion and Ci is the non-adsorbed protein
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concentration of the emulsion.
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2.7. Melt-down properties of ice cream
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The melt-down rate of ice creams were determined by the screen drip-through test (Goff and
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Hartel, 2013). The initial weight of the samples (at -20°C) were measured, placed on a mesh
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grid (2.5*2.5m) and allowed to stand in ambient temperature (25°C). The serum passing through
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the grid was collected and weight was recorded every 10 min over 90 min. Melt-down rates test
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was conducted in triplicate.
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2.8. Confocal laser scanning microscopy (CLSM)
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Confocal laser scanning microscopy (Leica Microsystem, Mannheim, Germany) with a 20 x
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objective lens was used to observe the microstructure of thawed ice cream emulsion. The fat
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phase was colored with Nile Red (NR) as per the method by Lent et al. (2008). 0.5 mL of 1 %
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NR in dimethysulfoxide and 0.5 mL 0.1% FITC in water was added to 500 g of ice cream mix.
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The stained sample was placed on a microscope slide and covered with a glycerol-coated cover
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slip. The samples were excited with a 488 nm Argon laser line and 633 nm line of Helium/Neon
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laser, and detected at 503-588 nm and 648-733nm, respectively.
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2.9. Statistical Analysis
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All experiments and associated measurements were performed at least in triplicate. Statistical
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analysis was performed using a two-way ANOVA (α < 0.05) by Statistix 9.0 (Statistix,
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Tallahassee, FL, USA).
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3.0. Results and Discussion
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3.1. Composition and properties of soy protein isolate and its hydrolysate
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The degree of hydrolysis of soy protein isolate and the emulsifying properties (EAI and ESI) of
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all samples are shown in Table 2. The EAI was increased by hydrolysis, mainly at the lowest DH
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(2.4%) for CSPI. Increased molecular flexibility and more exposed hydrophobic sites caused by
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hydrolysis led to better surface characteristics and functional properties. SPHPe emulsion had the
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highest stability index and SPHPa had the lowest stability index of all samples (Table 2). Fig. 1a
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shows the SDS-PAGE patterns of SPI with different degree of hydrolysis as raw ingredients. The
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protein profile of NSPI showed intact β-conglycinin subunits (α, α' and β) and glycinin (acidic
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and basic subunit) in the electrophoretic analysis (Fig.1a), which was consistent with the finding
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from previous research (Nishinari et al., 2014). Compared to NSPI, CSPI showed fading
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corresponding bands for β-subunit and basic subunits and no corresponding band to AB subunits
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in SDS-PAGE (Fig.1a), which was due to the loss during thermal treatment in production. The
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main subunits in SPHPe were α'-subunit, β-conglycinin, and basic subunit. The profile of SPHPa
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indicated all subunits from glycinin and β-conglycinin had been hydrolyzed and only the
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peptides with molecular weight less than 20 kDa were identified (Fig. 1a).
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The profiles of adsorbed protein on the fat globules in the thawed ice cream were also analyzed
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by SDS-PAGE (Fig.1b). All main subunits of β-conglycinin (α, α' and β) and glycinin (acidic
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and basic subunit) were identified in NSPI and CSPI samples except that CSPI samples did not
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have AB-subunit and basic subunit. β-conglycinin (α, α' and β) was present on the surface of
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SPHPe ice cream fat globules and the relative composition of β-subunit was the highest of all
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samples. SPHPa only had the peptides with low molecular weight of less than 20 kDa. The
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densitometric analysis of the main protein bands in SDS-PAGE are shown in Table 3. For NSPI,
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CSPI and SPHPe ice cream mix samples, the relative composition of α, α', acidic and basic
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subunit on the fat globules surface were less than those subunits in the ingredients. Acidic, α and
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α' subunit with a higher content of hydrophilic amino acids showed no strong tendency to be
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absorbed on the fat surface and can be displaced by monoglycerides from oil/water interface
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during aging (Chen, Liang, Li, & Chen, 2018; Nishinari, Fang, Guo, & Phillips, 2014). The
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relative composition of β-subunit indicated no difference in all ice cream samples between
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ingredients and adsorbed protein at water/oil interface. Keerati-u-rai & Corredig (2009) indicated
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β-subunits had a preferential adsorption to fat globules surface. β-subunit cannot be displaced by
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monoglycerides from oil/water interface during aging (Chen et al., 2018).
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3.2. Rheological properties of ice cream mixes
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The consistency index (K) and the flow behavior index (n) of ice cream mixes made with SPI
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and its hydrolysate are shown in Table 4. The flow behavior index (n) of all samples was below
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1, ranging from 0.46 to 0.58. All ice cream mixes exhibited non-Newtonian behavior, showing
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the apparent viscosity decreasing with increasing shear rate. SPHPa ice cream mixes had the
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lowest value in the flow behavior index (n), hence highest pseudoplastic properties. All polymer
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molecules including protein in all ice cream mixes tended to disentangle under shearing and
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align in the flow field, giving less resistance to flow (Innocente, Comparin, & Corradini, 2002).
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The flow properties of ice cream mixes were indicated by the consistency coefficient (K), which
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was attributed to the colloidal particles in the ice cream mix (Damodaran, 1997). In this study,
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the different protein sources impacted the consistency coefficient due to their molecule size,
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structure, composition and interaction with other protein molecules. The mix made with NSPI
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exhibited high K and apparent viscosity due to its compact molecular structure, requiring more
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shear stress to achieve the same shear rate. Interestingly, the mix made with SPHPe had the
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highest consistency coefficient in all samples. This was attributed to modification of SPI by
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pepsin, which increased the interaction between neighboring molecules due to more exposed
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hydrophobic and hydrophilic sites, conferring greater resistance to flow. CSPI mix consistency
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coefficient was not significantly different from SMP and SPHPa (p> 0.05), which means it had
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similar apparent viscosity at the same shear rate. The reason may be due to the removal of
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insoluble portion of SPI and the loss of subunits as discussed above. The molecular weight in
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SPHPa was below 20 kDa, which led to less protein polymerization and low consistency
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coefficients (K).
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3.3. Meltdown rate
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meltdown rates of ice cream with soy protein and its hydrolysates are shown in Fig.2. All ice
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cream samples in this study melted within 80 min. The ice cream made with SPHPe had the
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highest meltdown rate across all samples which ranged from 1.23% min-1 to 2.05 % min-1. The
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ice cream made with CSPI, SMP and SPHPa had the lowest melt rate, which was not
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significantly different from each other. Previous research indicates ice cream mix viscosity,
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overrun and ice crystals size influenced meltdown rate (Muse & Hartel, 2004; Sofjan & Hartel,
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2004). In this study, the protein concentration, overrun and freezing condition had been
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standardized to the same level in all treatment. The protein source was more dominant in
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affecting meltdown rate. For the mix viscosities, SPHPe mix had the highest viscosity (Table 4)
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but exhibited the highest meltdown rate, indicating that rheological properties of mix did not
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have a relationship with the meltdown rate. The fat partial coalescence and the formation of
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clumps of fat globules caused by destabilization was mainly related to ice cream meltdown rate
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through forming a three-dimensional network of fat globules, which attached and stabilized on
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the surface of air bubbles of ice cream (Cruz et al., 2009; Marshall, Goff, & Hartel, 2003; Muse
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& Hartel, 2004). Insufficient fat globules destabilization in ice cream resulted in a rapid
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meltdown rate (Goff & Hartel, 2013). The relationship between meltdown rate of ice cream and
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fat partial coalescence is discussed further in the section below.
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3.4.Particle Size Distribution
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The volume-weighted means (d4,3) and particle size distribution of the fat globules are good
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indicators of the development of fat coalescence (Méndez-Velasco & Goff, 2012). They were
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used to illustrate how the different degree of hydrolysis of soy protein isolate impacted on
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stabilization of the emulsions and fat globule coalescence after freezing ice cream mixes (Fig. 3).
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CSPI, SMP and SPHPa fresh mixes exhibited bimodal particle distribution with the first peak in
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the range of 0.1-10 um and the second peak 10-100 um. The main particle size distribution of
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NSPI and SPHPe fresh mixes were in range of 0.1-10 um with a long tail in the range of 10-100
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um. The particle distribution of SMP and CSPI mixes were in the range of 0.1-100 um with
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similar curves (Fig.4a). This indicated that SMP and CSPI had similar emulsifying properties in
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stabilizing the fresh ice cream mix, which agreed with the results of EAI and ESI (Table 2). Milk
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protein formed a film on the fat globules surface to prevent close contact via steric and
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electrostatic repulsion forces during homogenization (Méndez-Velasco & Goff, 2012). SPI and
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its hydrolysates aided the formation of stable emulsion via forming a physical barrier at the
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oil/water interface, which prevented the flocculation and coalescence of fat droplets (Li et al.,
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2016). After freezing, fat destabilization led to an increase in volume of larger particle sizes with
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increasing second peak in the range of 10-100 um, representing partial coalescence of emulsion
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droplets compared to the fresh emulsion for all samples (Fig.4b) (Boode & Walstra, 1993). The
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SPHPa thawed sample showed the highest volume in the second peak, followed by SMP and
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CSPI thawed samples. The larger particles in the thawed sample of SPHPe was lowest among all
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samples, indicating less fat globule coalescence (Fig.4b). d4,3 of SPHPa and SMP showed no
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significant difference in the fresh mix and the thawed ice cream mix (Fig 3). The particle size
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(d4,3) of fat globules in SMP ice cream in this study was in accordance with the finding by Sung
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& Goff (2010). All thawed samples had a significantly increased d4,3 compared to the fresh
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samples with exception of SPHPe (Fig.3). SPHPe sample had the smallest increase in d4,3
302
amongst all samples, from 2.95 um to 4.69 um. The highest fat globules aggregation occurred in
303
samples made with SPHPa, where d4,3 increased from 1.99 um to 9.45 um. d4,3 of thawed ice
304
cream made with CSPI and NSPI were not significantly different from SMP, indicating similar
305
fat globules partial coalescence was formed during freezing compared to SMP. The degree of fat
306
destabilization was controlled by the properties of the fat crystal and the adsorbed protein at the
307
oil-water interface (Segall & Goff, 2002; Vanapalli, Palanuwech, & Coupland, 2002). Unsalted
308
butter used as the fat source consisted of high and low melting point fat triacylglycerol, existing
309
as a mixture of 2/3 crystalline and 1/3 liquid at 4°C (Adleman & Hartel, 2001), which ensured
310
the occurrence of partial coalescence with desired fat aggregated structure (Sung & Goff, 2010).
311
The mechanical properties of adsorbed protein film at oil/water interface and steric repulsion
312
between protein molecules prevented fat globules from coalescences in the aqueous phase (Bos
313
& Vliet, 2001). Most proteins used as the surfactant at oil/water interfaces decrease surface
314
tension about 15-20 mNm-1 but small surfactants can lower by 30-40 mNm-1 (Damodaran, 2005).
315
The displacement of adsorbed protein by emulsifier occurs because emulsifier has lower
316
interfacial tension at water/oil interface. As result, the weakened layer provides the condition for
317
the fat globules destabilization in dynamic freezing (Boode & Walstra, 1993; Gelin, Poyen,
318
Courthaudon, Le , & Lorient, 1994; Pelan, Watts, Campbell, & Lips, 1997). In this study, 0.1%
319
monoglycerides displaced soy protein and its hydrolysates from the fat globules surface during
320
mix aging except SPHPe. The high percentage of β-subunit in SPHPe kept the ice cream
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emulsion stable and cannot be displaced by monoglycerides. The quantities of displaced protein
322
and the profile change of protein at oil/water interface for all samples have been discussed in
323
Section 3.1 and further in 3.5.
324
3.5. Adsorbed protein fraction and composition
325
The extent of fat globule partial coalescence in ice cream mix has a direct relationship with the
326
adsorbed protein layer at oil/water interface (Bolliger et al., 2000). Measuring the change in
327
adsorbed milk protein amount at the oil/water surface in fresh ice cream mix and thawed ice
328
cream was used to predict the occurrence of partial coalescence during freezing (Barfod, Krog,
329
Larsen, & Buchheim, 1991; Gelin et al., 1994; Lai, O'Connor, & Eyres, 2006; Pelan et al., 1997).
330
Fig.5 shows the adsorbed protein fraction of the SMP, NSPI, CSPI, SPHPe and SPHPa at
331
oil/water in the fresh ice cream mixes and thawed ice cream mixes. Intact soy protein such as
332
NSPI and CSPI exhibited lower adsorbed amount at the interface compared to SPHPe and
333
SPHPa, which were attributed to the large molecule size and less molecule structure flexibility.
334
Limited enzymatic hydrolysis improved soy protein molecular flexibility and achieved better
335
emulsifying properties (Li et al., 2016). During ice cream aging, more protein desorption by
336
monoglycerides may lower steric stabilization of the fat globules to creates a thinner film, which
337
renders it prone to partial coalescence during ice cream freezing. After aging, the fraction of
338
absorbed protein on the fat globules decreased in all ice cream mix. The displaced fractions of
339
protein in ice cream mixes in this study were in the range of 8.0% to 22.6%. The largest fraction
340
of protein at the fat globules displaced during aging and freezing was SPHPa mix (22.6%),
341
which was comparable to that of SMP (21.8%). The lowest fraction displacement was SPHPe
342
mix (8.0%). Gelin et al. (1994) indicated milk protein can be displaced by monoglycerides from
343
oil/water interface during ice cream mix aging due to formation of favorable interface. For soy
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protein, all subunits and peptides can be displaced by monoglycerides except β–subunit due to its
345
strong adsorption ability in oil/water interface (Fig. 1b). Hence the protein composition absorbed
346
on fat globules indicated the emulsifier functionality at oil-water interface and the degree of
347
steric stabilization of fat globules. Pelan et al. (1997) found the emulsion stabilized by whey
348
proteins exhibited higher partial coalescence compared to skim milk powder because the
349
adsorbed layer formed by whey protein molecules was not as thick as casein micelles. For NSPI,
350
CSPI, SPHPa and SMP mixes, a large amount protein was displaced by monoglycerides, which
351
led to a lower steric stabilization of the droplet and rendered it to be more prone to partial
352
coalescence (Fig.3). SPHPa displayed an intermediate adsorbed protein load (Fig. 5), a high fat
353
particle size after freezing and thawing (Fig. 3) and consequently a low meltdown rate (Fig. 2),
354
all similar to SMP (Fig. 2). SPHPe with high percentage of β-subunit kept the emulsion stable
355
and only small portion of protein was displaced by monoglycerides (Fig.5), resulting in less
356
partial coalescence. (Fig. 3) and high meltdown rate (Fig. 2). In this case, since there was
357
insufficient partial coalescence of fat globules to stabilize the aqueous lamella between air cells
358
and prevent drainage, the serum inside the ice cream structure drained at a faster rate. Two
359
mechanisms were given to describe the mechanism of the displacement. Hoffmann & Reger
360
(2014) reported surfactants interacted with protein molecules at oil/water interface, resulting in
361
increased protein hydrophilic properties and desorbing from the interface. Another mechanism
362
was that emulsifiers were adsorbed into localized protein film defects, accumulating and
363
developing a domain, which forced weak adsorbed surface proteins towards aqueous phase
364
(Mackie et al., 2003). However, further study for the displacement mechanism of soy protein
365
molecules and monoglycerides at oil/water interface is advisable.
366
3.6. Confocal Laser scanning microscopy (CLSM)
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Confocal laser scanning microscopy (CLSM) was used to visually examine the structure of fat
368
globules in the melted ice cream mixes. The fat globules of thawed ice cream mixes made with
369
SMP, NSPI, CSPI and SPHPa interact with each other to form larger particles at different degree,
370
indicating partial coalescence of the milkfat globules compared to the fresh samples (Fig.6).
371
Clumps of fat globules were present in the serum phase, indicated by the intense red regions in
372
the micrographs. Partially coalesced fat globules covered the air interface incompletely and
373
stabilized air bubble in ice cream structure due to its hydrophobicity (Koxholt, Eisenmann, &
374
Hinrichs, 2001). Fig.6 demonstrates that there was a lot of fat agglomeration in the micrograph
375
of ice cream with SPHPa, similar to that shown by light scattering in Fig.3, which suggests a
376
potential alternative protein source in making ice cream. There are few fat globule aggregates
377
formed in ice cream made with SPHPe due to its stable emulsion.
378
4.0. Conclusion
379
This study presents new information on the role of soy protein isolate and its hydrolysates in ice
380
cream structure formation, including soy protein composition profile at the fat interface and ice
381
cream physico-chemical properties. The composition of soy protein isolate had a significant
382
impact on the degree of fat destabilization and melt-down rate of the ice cream. The relatively
383
high proportion of β-subunits in SPHPe prevented the occurrence of sufficient fat globules
384
partial coalescence and had the highest melting rate in all samples. The displacement of adsorbed
385
α', α, acidic and basic subunit in CSPI, NSPI and SPHPa on the fat globules by monoglycerides
386
promoted formation of fat partial coalescence and contributed to ice cream microstructure with
387
desirable melting properties. The meltdown and rheological properties of ice cream made with
388
SPHPa were comparable to SMP, which suggests a potential alternative to SMP for ice cream
389
manufacture.
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Conflict of interest
391 392
The authors state that there are no conflicts of interest regarding publication of this article.
393
Acknowledgement
394
This work was supported by the National Natural Science Foundation of China (Grant No.
395
31471583) and National First-class discipline program of Food Science and Technology (Grant
396
No. JUFSTR20180201).
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397
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Figure Captions
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Fig. 1. SDS-PAGE patterns of soy protein and its hydrolysates (a-ingredient, b- absorbed protein on the fat globules surface). BS: basic subunits; AS: acidic subunits; AB: their aggregates. α, α' and β are subunits from β-conglycinin.
536 537 538
Fig. 2. Meltdown rate of ice cream. Values are means ± standard deviation (the error between multiple measurements); values with same superscript letter are not significantly different (p > 0.05).
539 540 541
Fig. 3. The volume weighted mean particle diameter (d4,3) of ice cream with different soy protein isolate. Values are means ± standard deviation (the error between multiple measurements); values with same superscript letter are not significantly different P > 0.05.
542
Fig. 4. Droplet size distribution of (a) fresh ice cream mix and (b) thawed ice cream
543 544
Fig. 5. Absorbed protein fraction at the surface of fat globules. Values are means ± standard deviation (the error between multiple measurements)
545 546 547
Fig. 6. Microstructure of (a) fresh ice cream mix and (b) thawed ice cream with SMP, NSPI, CSPI, SPHPe and SPHPa. Bar r = 25 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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ACCEPTED MANUSCRIPT Table 1 Formulations of ice cream mixes containing different protein Ingredients
SMP
NSPI
CSPI
SPHPe
SPHPa
(%)
(%)
(%)
(%)
(%)
10.00
10.00
10.00
10.00
10.00
Fat
10.00
10.00
10.00
10.00
10.00
CSS
4.00
4.00
4.00
4.00
4.00
SMP
10.00
0
0
0
0
NSPI
0
4.40
0
CSPI
0
0
4.30
SPHPe
0
0
0
SPHPa
0
0
0
Lactose
0
5.60
5.70
Stabilizers
0.15
0.15
0.15
Monoglycerides
0.15
0.15
0.15
IW
65.70
65.70
65.70
100
100
100
a
0
0
0
5.15
0
0
4.50
4.85
5.50
0.15
0.15
0.15
0.15
65.70
65.70
100
100
SC
0
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Total
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Sugar
Abbreviations: NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate; SPHPe, Soy Proteins
Isolate hydrolyzed by Pepsin; SPHPa: Soy protein Isolate hydrolyzed by Papain; CSS, Corn Syrup Solid; IW:
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Ingredient Water.
ACCEPTED MANUSCRIPT Table 2 Properties of soy protein isolate and its hydrolysates EAI (m2/g)
DH
ESI (%)
SMP
-
33.96 ± 0.32c
85.06 ± 0.51b
NSPI
0.0% ± 0.1a
36.69 ± 1.04b
89.63 ± 1.20ab
CSPI
2.4% ± 0.2b
32.65 ± 0.16c
86.68 ± 4.65b
SPHPe
3.6% ± 0.4c
42.21 ± 0.99a
94.72 ± 0.88a
SPHPa
15.6% ± 0.7d
40.43 ± 0.72a
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Ingredients
63.20 ± 0.81c
*DH: Degree of hydrolysis; EAI: Emulsifying activity index; ESI: emulsion stability index. Values with the same superscript letters within each column are not significantly different (P>0.05). DH is not tested for SMP.
SC
Abbreviations: SMP, Skim milk powder; NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate;
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SPHPe, Soy Proteins Isolate hydrolyzed by Pepsin; SPHPa, Soy Protein Isolate hydrolyzed by Papain.
ACCEPTED MANUSCRIPT Table 3 The densitometric analysis: relative compositions (%) of soy proteins and hydrolysates in both the starting ingredient materials and adsorbed to the fat globules as isolated from melted ice cream α'-
Subunit
α
β
AB
AS
BS
11.5
6.1
Ingredient
5.5
6.3
11.2
24.9
Adsorbed protein
4.5
5.4
10.8
27.0
Ingredient
9.5
13.1
4.1
0.0
Adsorbed Protein
6.5
6.6
3.9
0.0
Ingredient
25.4
8.7
14.3
Adsorbed Protein
10.1
7.0
13.9
Ingredient
0.0
0.0
0.0
Adsorbed protein
0.0
0.0
0.0
CSPI
5.3
21.5
0.0
5.8
0.0
0.0
4.1
17.9
0.0
0.0
4.2
0.0
0.0
0.0
0.0
0.0
0.0
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SPHPa
7.8
SC
SPHPe
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NSPI
*Abbreviations: NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate; SPHPe, Soy Protein Isolate hydrolyzed by Pepsin; SPHPa, Soy Protein Isolate hydrolyzed by Papain. BS: basic subunits; AS: acidic
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ACCEPTED MANUSCRIPT Table 4 Rheological properties of ice cream mixes Herschel-Bulkley Parameter K (Pa Sn)
Apparent Viscosity (Pa s)
n
τ0
10 s-1
100 s-1
SMP
0.64±0.24b
0.58±0.03ab
0.19±0.05a
0.26
0.09
NSPI
1.23±0.43b
0.55±0.03a
0.35±0.09a
0.47
0.16
CSPI
b
0.74±0.22
0.49±0.06
a
0.24
0.07
SPHPe
2.12±0.96a
0.55±0.05ab
SPHPa
b
0.46±0.03
c
0.15±0.09
0.72±0.39a a
0.32±0.01
RI PT
0.86±0.03
bc
0.82
0.27
0.28
0.07
* K = consistency index; n = flow behavior index; τ0 = Yield Stress. Values with the same superscript letters within each column are not significantly different (P>0.05). Abbreviations: SMP, Skim milk powder; NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate; SPHPe, Soy Proteins Isolate hydrolyzed by Pepsin; SPHPa,
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SPHPe
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2. The interaction between SPI subunits and emulsifier decided meltdown properties.
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3. SPHPe ice cream show insufficient fat partial-coalescence and rapid melting rate.
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4. Ice cream with SPHPa exhibited the similar melting property as milk protein.
S0268-005X(18)32290-2
DOI:
https://doi.org/10.1016/j.foodhyd.2019.02.045
Reference:
FOOHYD 4971
To appear in:
Food Hydrocolloids
Received Date: 22 November 2018 Revised Date:
19 February 2019
Accepted Date: 25 February 2019
Please cite this article as: Chen, W., Liang, G., Li, X., He, Z., Zen, M., Gao, D., Qin, F., Goff, H.D., Chen, J., Effects of soy proteins and hydrolysates on fat globule coalescence and meltdown properties of ice cream, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/j.foodhyd.2019.02.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Protein
Soy
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Graphical abstract
Isolate
pepsin (SPHPe)
protein
hydrolyzed
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papain (SPHPa)
Commercial Soy Protin (CSPI)
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Isolate
Composition and properties of soy protein isolate and its hydrolysate
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The interaction between soy protein subunits and monoglycerides at oil-water interface
protein
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(NSPI)
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Wenpu Chenab , Guijiang Liangab, XiangLic, Zhiyong Heab ,Maomao Zenab ,Daming Gaoab, Fang
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Qin ab, H. Douglas Goffd, Jie Chen*ab
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a
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China
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International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, PR, China
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School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR, China
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Department of Food Science, University of Guelph, ON N1G2W1, Canada
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Effects of soy proteins and hydrolysates on fat globule coalescence and meltdown properties of ice cream
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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR.
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*Corresponding author:
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Professor Jie Chen
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State Key Laboratory of Food Science and Technology, School of Food Science and Technology,
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Jiangnan University, Wuxi, Jiangsu, 214122, China
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Tel.: +86-510-85329032
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Fax: +86-510-85919065
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E-mail address: [email protected]
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Abstract
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The effect of soy protein isolate and its hydrolysates on ice cream mix stability and melt-down
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properties of ice cream were investigated. Ice creams were made with 10% milk fat, 3.5%
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protein and 34.3% total solids, and all contained 0.15% added monoglycerides. The proteins
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used were native soy protein isolate (NSPI), commercial soy proteins isolate (CSPI), soy protein
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hydrolyzed by pepsin (SPHPe), soy protein hydrolyzed by papain (SPHPa) and skim milk
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powder (SMP). Ice cream with SPHPe containing the highest relative composition of β-subunit
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showed good mix emulsion stability and rapid melting rate because β-subunit cannot be
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displaced by the monoglycerides, leading to lack of fat partial coalescence in the ice cream.
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SPHPa ice cream exhibited comparable functionality to SMP in rheological and meltdown
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properties. SDS-PAGE results indicated that α subunit, α' subunit, acidic subunit, basic subunit
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and small molecule polypeptide were displaced by monoglycerides during ice cream aging.
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Selective hydrolysis of soy protein can be used for ice cream with sufficient fat partial
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coalescence and good melt-down rates.
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Key Word： Soy Protein Isolate, Hydrolysates, Ice Cream
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Introduction
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Ice cream is a complex food emulsion system with ice crystals, dispersed fat globules and air
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cells, protein-hydrocolloid structures and a continuous unfrozen aqueous phase. A higher degree
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of fat destabilization (a high level of partially coalesced fat globule clusters) stabilizes the air
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phase and contributes to the physical attributes of ice cream such as texture and melting
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properties (Goff & Hartel, 2013). Traditionally, milk protein is a major source in making ice
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cream due to its multiple desirable functionalities including stabilizing fat droplets by steric
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repulsion, interaction with emulsifiers at the interface, stabilizing air bubbles and enhancing
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unfrozen phase viscosity to prevent the growth of larger ice crystals besides its nutritional
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function (Dickinson, 2003; Goff & Hartel, 2013). The role of milk protein in ice cream has been
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fully explored in shape retention, fat globule destabilization and melting properties (Alvarez,
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Wolters, Vodovotz, & Ji, 2005; Daw & Hartel, 2015; Patel, Baer, & Acharya, 2006).
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Soy protein is considered as an alternative to or extension of milk protein in food systems due to
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its availability, emulsifying properties, plant-derived source and health benefits (Abdullah et al.,
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2003; Anderson, Smith, & Washnock, 1999; Biswas, Chakraborty, & Choudhuri, 2002). As a
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macromolecular emulsifier with amphiphilic nature, soy protein isolate stabilizes oil-water or air-
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water interfaces by forming a viscoelastic film at the oil/water interface (Palazolo, Mitidieri, &
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Wagner, 2003; Puppo et al., 2008). The characteristics of soy protein isolate such as solubility
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and emulsification make it possible for it to be used in ice cream (Dervisoglu, Yazici, &
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Aydemir, 2005; Friedeck, Aragul-Yuceer, & Drake, 2003). The addition of soy protein isolate in
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ice cream reduces the effect of heat shock on ice recrystallization and melting rate (Pereira et al.,
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2011). Replacing partial skim milk powder with soy extract in ice cream achieved smaller ice
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crystals and minimized ice recrystallization under temperature fluctuations (Pereira et al., 2011).
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However, 100% soy protein isolate as the protein source in the formula led to high viscous mix
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and meltdown-resistant ice cream (Akesowan, 2009; Dervisoglu et al., 2005). There has been
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some published research about soy protein functionality in the formation of ice cream structure.
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Soy protein with larger molecular weight and compact structure had limited surface activity at
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the oil/water interface due to slow rate of diffusion and adsorption (Santiago et al., 2008), which
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may impact the formation of partial fat coalescence during ice cream freezing. The production of
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high-quality ice cream with soy protein would therefore require comparable ice cream mix
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viscosity as milk protein and the formation of partial fat coalescence, which are necessary for a
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stable foam structure in ice cream, shape retention dryness and slowness of meltdown (Goff,
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1997). Optimization of functionalities of soy protein structure is of interest to achieve a high
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degree of fat globule partial coalescence in ice cream making.
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Limited enzymatic hydrolysis of soy protein has been used to expose concealed hydrophobic
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groups and increase molecular flexbility, which indicates good affinity at the oil/water interface
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(Conde & Patino, 2007; Panyam & Kilara, 1996). Huang, Catignani, & Swaisgood (1996)
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reported that limited enzymatic hydrolysis with pepsin increased emulsifying activity index and
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achieved smaller fat globules compared to intact soy protein. The objective of this study was to
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apply pepsin and papain to modify soy protein for hydrolysates with different viscoelastic and
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interfacial properties and identify the main soy protein composition that may impact ice cream
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microstructure and meltdown properties. For this purpose, ice cream and its mixes formulated
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with different soy protein hydrolysates were characterized by rheology, particles size distribution,
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the amount and peptide profile of adsorbed protein at oil/water interface, meltdown rate and
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structure by confocal microscopy.
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2.0. Materials and methods
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2.1. Materials
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Soy bean was obtained from local market (Wuxi, China). Soy protein isolate (SPI) was prepared
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according to the procedure given by Diftis & Kiosseoglou (2006). Kjeldahl method (N x 6.25)
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was used to determine protein content of the samples (Ryan et al., 2008). Commercial soy
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protein isolate (CSPI) were obtained from Solae, USA. Skim milk powder (SMP), unsalted
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butter (Anchor) and lactose were obtained from Fonterra, New Zealand. The main composition
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of SMP includes 35% milk protein, minerals, lactose, and less than 1% fat content. Corn syrup
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solids (36DE) was obtained from Cargill, China. Guar gum, locust beam guar and
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monoglycerides were obtained from Danisco, China. Pepsin (3000 U/mg) was purchased from
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Sangon Biotech, Shanghai, China and papain (2000 U/mg) was purchased from Regal, Shanghai,
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China. Deionized water was used as the ingredient water and all other chemicals used were
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analytical grade unless otherwise specified.
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2.2. Preparation and characterization of soy protein isolate and its hydrolysates
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Commercial soy protein isolate (CSPI) was reconstituted and centrifuged to remove the insoluble
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portion (320 x g, 5 min, 25°C). The supernatant was freeze-dried for powder. For native soy
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protein isolate and its hydrolysates, defatted soy protein flakes were dispersed in a 9-fold weight
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of distilled water at 25°C, and pH value of the slurry was adjusted to 8.0 with 2M NaOH. The
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slurry was stirred for 120 min and centrifuged (10000 x g, 20 min, 25°C) to remove the insoluble
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portion. The supernatant was adjusted to pH 4.5 with 2M HCl and acid-precipitated soy protein
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curd was collected by centrifugation (3300 x g, 10 mins, 25°C). Three portions of acid-
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precipitated soy protein curd were prepared. The first portion was made as native soy protein
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isolate (NSPI). The precipitate was dispersed in a 4-fold weight of water and neutralized to pH
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7.0 with 2M NaOH before freeze drying.
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The second portion of curd was dispersed in water to the final concentration 5% (w/v) at 40°C
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and pH was adjusted to 2.0 with 2M HCl. Pepsin was added to the SPI dispersion with enzyme-
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to-SPI ratio of 0.3 wt% with stirring. The slurry was incubated at 40°C for 2h and was
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terminated by the heat treatment (120 °C, 15s). The collected precipitate was dispersed in a 4-
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fold weight of distilled water and neutralized to pH 7.0 with 2M NaOH before freeze drying. It
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will be referred to hereafter as soy proteins hydrolyzed by pepsin (SPHPe). The third portion of
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curd was dispersed in water to the concentration 5% (w/v) and adjusted to pH 7.0 with NaOH.
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0.5 wt% papain was added and incubated for 30 min at 50°C. The reaction was terminated by
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heat treatment (120°C, 15s). The solution was freeze-dried to obtain soy proteins hydrolyzed by
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papain (SPHPa). The protein content in NSPI, CSPI, SPHPe and SPHPa are 79.5%, 81.4%, 68.0%
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and 77.8%. The rest of the main composition in NSPI, CSPI, SPHPe and SPHPa are soluble
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carbohydrate, minerals and very small amount of soy oil residue.
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The degree of hydrolysis (DH) was determined by TNBS method described by Adler-Nissen
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(1979). Emulsifying activity index (EAI) and emulsion stability index (ESI) of the samples were
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determined by the method described by Guo et al. (2015).
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The polypeptide profiles of soy protein isolate and its hydrolysates were determined by sodium
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dodecyl sulphate-polyarylamide gel electrophoresis (SDS-PAGE) according to the procedure
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given by Li et al. (2016) and Keerati-u-rai & Corredig (2009), with slight modification. A mini-
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protein electrophoresis system (Bio-Rad Laboratories, Hercules, CA, U.S.A.) was used for
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analysis. For the raw ingredients, soy protein isolate and its hydrolysates were dissolved in the
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buffer (0.0625M of Tris-HCl, 10% glycerin, 2% SDS and 0.0025% bromophenol blue) to 1 mg/
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mL protein solution. After heating for 3 min in boiling water and centrifuging at 5000 x g for 10
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min at room temperature with a TGL-16G 144 centrifuge (Anting Scientific Instrument Co. Ltd.,
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Shanghai, China), aliquots (20 µL) of the prepared samples were loaded onto the gels. For
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adsorbed protein on the fat globules in the thawed ice creams, the mixes were centrifuged at
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10000g for 45 min at 25 °C in a temperature-controlled centrifuge (SIGMA 3K15, SIXMA,
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Germany). Cream was collected and was dried on the filter paper. Collected cream was re-
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suspended with ultrapure water to the initial volume then mixed with equal amount of buffer for
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composition determination using electrophoresis. Coomassie Brilliant Blue (G-250) was used to
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stain the gel. All gels were scanned by a computing densitometer (Molecular Imager
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ChemiDocXRS+, Bio-Rad, USA) Image Lab software (Bio-Rad, USA) was used to integrate the
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intensities of bands.
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2.3. Formulation of ice cream and processing
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Ice cream mixes were prepared with the formulation in Table 1 with the same protein content
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(3.5% w/w). Each ice cream mix was pasteurized at 85 °C for 1 min. The mix subsequently went
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through 2- stage homogenizer (17.2/3.4 MPa) (NANO Homogenize Machine, Model AH-Basic,
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ATS Engineering Inc). The mix was immediately cooled to 4°C and aged for 24h at 4°C. Ice
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cream was produced in batch freezer (Model 104, Taylor Freezers, Rockton, IL, USA) with
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outlet temperature between - 5 and - 6 °C. The overrun of ice cream was in the range of 40-50%.
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Ice creams were collected in paper containers with weight around 90-100 g and hardened below
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-30 °C in freezer for at least 72h before further testing.
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2.4. Rheological properties
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The rheological profile of all ice cream mixes were measured in a HAAKE MARS Rheometer
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(Thermo Scientific, Germany) equipped with cone and plate (40 mm, 2°). Sample size was 2 mL.
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Samples were presheared at 20 s-1 for 30s and the shear stress was recorded at shear rate from 0s-1
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to 300s-1 over 420s then a decreasing shear rate to 0s-1 over 420s. Rheological data were
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modelled according to Herschel-Bulkley model. τ =τ0 + K • n
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where τ is shear stress, K is consistency coefficient, γ∙ is shear rate, n is flow behavior index and
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τ0 is the yield stress.
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2.5. Particle size distribution
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Particle size distribution of fresh and melted ice cream mixes were determined by Microtrac
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BlueWave S3500 Laser Diffraction Particle Size Analyzer (Microtrac Inc. Montgomeryville, PA,
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USA). Samples were directly diluted (1:1000) in the sample chamber with MiliQ water at 16-
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18°C. The refractive index of the emulsion was taken as 1.33. Measurements were performed at
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ambient temperature and repeated in triplicate. Volume weighted mean (d4,3) were recorded.
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2.6. Adsorbed protein fraction determination
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The fraction of adsorbed protein (AP %) of fresh and thawed samples were determined using the
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method described by Ye (2008) and Chen et al. (2019) with slight modification. Each sample
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(4mL) was centrifuged at 10000g for 45 min at 25 °C in a temperature-controlled centrifuge
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(SIGMA 3K15, SIXMA, Germany). After the centrifugation, the aqueous layer was carefully
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removed using a syringe. The cream layer was re-dispersed in deionized water and centrifuged at
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10000g for 45 min at 25 °C. The subnatants were filtered through 0.22 um filters (Milipore
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Corp.). Kjeldahl method (N x 6.25) (KDN-103F, Automatic Nitrogen Determinator, Qianjian,
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Shanghai, China) was used to determine the filtrates and the total protein in the mix. Adsorbed
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protein fraction (%) was calculated by the formula below:
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Adsorbed protein fraction (%) = (C0- Ci)/C0 x 100%
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where C0 is the initial protein concentration of the emulsion and Ci is the non-adsorbed protein
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concentration of the emulsion.
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2.7. Melt-down properties of ice cream
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The melt-down rate of ice creams were determined by the screen drip-through test (Goff and
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Hartel, 2013). The initial weight of the samples (at -20°C) were measured, placed on a mesh
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grid (2.5*2.5m) and allowed to stand in ambient temperature (25°C). The serum passing through
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the grid was collected and weight was recorded every 10 min over 90 min. Melt-down rates test
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was conducted in triplicate.
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2.8. Confocal laser scanning microscopy (CLSM)
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Confocal laser scanning microscopy (Leica Microsystem, Mannheim, Germany) with a 20 x
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objective lens was used to observe the microstructure of thawed ice cream emulsion. The fat
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phase was colored with Nile Red (NR) as per the method by Lent et al. (2008). 0.5 mL of 1 %
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NR in dimethysulfoxide and 0.5 mL 0.1% FITC in water was added to 500 g of ice cream mix.
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The stained sample was placed on a microscope slide and covered with a glycerol-coated cover
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slip. The samples were excited with a 488 nm Argon laser line and 633 nm line of Helium/Neon
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laser, and detected at 503-588 nm and 648-733nm, respectively.
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2.9. Statistical Analysis
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All experiments and associated measurements were performed at least in triplicate. Statistical
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analysis was performed using a two-way ANOVA (α < 0.05) by Statistix 9.0 (Statistix,
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Tallahassee, FL, USA).
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3.0. Results and Discussion
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3.1. Composition and properties of soy protein isolate and its hydrolysate
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The degree of hydrolysis of soy protein isolate and the emulsifying properties (EAI and ESI) of
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all samples are shown in Table 2. The EAI was increased by hydrolysis, mainly at the lowest DH
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(2.4%) for CSPI. Increased molecular flexibility and more exposed hydrophobic sites caused by
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hydrolysis led to better surface characteristics and functional properties. SPHPe emulsion had the
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highest stability index and SPHPa had the lowest stability index of all samples (Table 2). Fig. 1a
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shows the SDS-PAGE patterns of SPI with different degree of hydrolysis as raw ingredients. The
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protein profile of NSPI showed intact β-conglycinin subunits (α, α' and β) and glycinin (acidic
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and basic subunit) in the electrophoretic analysis (Fig.1a), which was consistent with the finding
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from previous research (Nishinari et al., 2014). Compared to NSPI, CSPI showed fading
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corresponding bands for β-subunit and basic subunits and no corresponding band to AB subunits
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in SDS-PAGE (Fig.1a), which was due to the loss during thermal treatment in production. The
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main subunits in SPHPe were α'-subunit, β-conglycinin, and basic subunit. The profile of SPHPa
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indicated all subunits from glycinin and β-conglycinin had been hydrolyzed and only the
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peptides with molecular weight less than 20 kDa were identified (Fig. 1a).
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The profiles of adsorbed protein on the fat globules in the thawed ice cream were also analyzed
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by SDS-PAGE (Fig.1b). All main subunits of β-conglycinin (α, α' and β) and glycinin (acidic
221
and basic subunit) were identified in NSPI and CSPI samples except that CSPI samples did not
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have AB-subunit and basic subunit. β-conglycinin (α, α' and β) was present on the surface of
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SPHPe ice cream fat globules and the relative composition of β-subunit was the highest of all
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samples. SPHPa only had the peptides with low molecular weight of less than 20 kDa. The
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densitometric analysis of the main protein bands in SDS-PAGE are shown in Table 3. For NSPI,
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CSPI and SPHPe ice cream mix samples, the relative composition of α, α', acidic and basic
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subunit on the fat globules surface were less than those subunits in the ingredients. Acidic, α and
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α' subunit with a higher content of hydrophilic amino acids showed no strong tendency to be
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absorbed on the fat surface and can be displaced by monoglycerides from oil/water interface
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during aging (Chen, Liang, Li, & Chen, 2018; Nishinari, Fang, Guo, & Phillips, 2014). The
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relative composition of β-subunit indicated no difference in all ice cream samples between
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ingredients and adsorbed protein at water/oil interface. Keerati-u-rai & Corredig (2009) indicated
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β-subunits had a preferential adsorption to fat globules surface. β-subunit cannot be displaced by
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monoglycerides from oil/water interface during aging (Chen et al., 2018).
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3.2. Rheological properties of ice cream mixes
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The consistency index (K) and the flow behavior index (n) of ice cream mixes made with SPI
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and its hydrolysate are shown in Table 4. The flow behavior index (n) of all samples was below
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1, ranging from 0.46 to 0.58. All ice cream mixes exhibited non-Newtonian behavior, showing
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the apparent viscosity decreasing with increasing shear rate. SPHPa ice cream mixes had the
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lowest value in the flow behavior index (n), hence highest pseudoplastic properties. All polymer
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molecules including protein in all ice cream mixes tended to disentangle under shearing and
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align in the flow field, giving less resistance to flow (Innocente, Comparin, & Corradini, 2002).
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The flow properties of ice cream mixes were indicated by the consistency coefficient (K), which
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was attributed to the colloidal particles in the ice cream mix (Damodaran, 1997). In this study,
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the different protein sources impacted the consistency coefficient due to their molecule size,
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structure, composition and interaction with other protein molecules. The mix made with NSPI
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exhibited high K and apparent viscosity due to its compact molecular structure, requiring more
248
shear stress to achieve the same shear rate. Interestingly, the mix made with SPHPe had the
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highest consistency coefficient in all samples. This was attributed to modification of SPI by
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pepsin, which increased the interaction between neighboring molecules due to more exposed
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hydrophobic and hydrophilic sites, conferring greater resistance to flow. CSPI mix consistency
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coefficient was not significantly different from SMP and SPHPa (p> 0.05), which means it had
253
similar apparent viscosity at the same shear rate. The reason may be due to the removal of
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insoluble portion of SPI and the loss of subunits as discussed above. The molecular weight in
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SPHPa was below 20 kDa, which led to less protein polymerization and low consistency
256
coefficients (K).
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3.3. Meltdown rate
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meltdown rates of ice cream with soy protein and its hydrolysates are shown in Fig.2. All ice
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cream samples in this study melted within 80 min. The ice cream made with SPHPe had the
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highest meltdown rate across all samples which ranged from 1.23% min-1 to 2.05 % min-1. The
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ice cream made with CSPI, SMP and SPHPa had the lowest melt rate, which was not
264
significantly different from each other. Previous research indicates ice cream mix viscosity,
265
overrun and ice crystals size influenced meltdown rate (Muse & Hartel, 2004; Sofjan & Hartel,
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2004). In this study, the protein concentration, overrun and freezing condition had been
267
standardized to the same level in all treatment. The protein source was more dominant in
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affecting meltdown rate. For the mix viscosities, SPHPe mix had the highest viscosity (Table 4)
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but exhibited the highest meltdown rate, indicating that rheological properties of mix did not
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have a relationship with the meltdown rate. The fat partial coalescence and the formation of
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clumps of fat globules caused by destabilization was mainly related to ice cream meltdown rate
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through forming a three-dimensional network of fat globules, which attached and stabilized on
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the surface of air bubbles of ice cream (Cruz et al., 2009; Marshall, Goff, & Hartel, 2003; Muse
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& Hartel, 2004). Insufficient fat globules destabilization in ice cream resulted in a rapid
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meltdown rate (Goff & Hartel, 2013). The relationship between meltdown rate of ice cream and
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fat partial coalescence is discussed further in the section below.
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3.4.Particle Size Distribution
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The volume-weighted means (d4,3) and particle size distribution of the fat globules are good
279
indicators of the development of fat coalescence (Méndez-Velasco & Goff, 2012). They were
280
used to illustrate how the different degree of hydrolysis of soy protein isolate impacted on
281
stabilization of the emulsions and fat globule coalescence after freezing ice cream mixes (Fig. 3).
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CSPI, SMP and SPHPa fresh mixes exhibited bimodal particle distribution with the first peak in
283
the range of 0.1-10 um and the second peak 10-100 um. The main particle size distribution of
284
NSPI and SPHPe fresh mixes were in range of 0.1-10 um with a long tail in the range of 10-100
285
um. The particle distribution of SMP and CSPI mixes were in the range of 0.1-100 um with
286
similar curves (Fig.4a). This indicated that SMP and CSPI had similar emulsifying properties in
287
stabilizing the fresh ice cream mix, which agreed with the results of EAI and ESI (Table 2). Milk
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protein formed a film on the fat globules surface to prevent close contact via steric and
289
electrostatic repulsion forces during homogenization (Méndez-Velasco & Goff, 2012). SPI and
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its hydrolysates aided the formation of stable emulsion via forming a physical barrier at the
291
oil/water interface, which prevented the flocculation and coalescence of fat droplets (Li et al.,
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2016). After freezing, fat destabilization led to an increase in volume of larger particle sizes with
293
increasing second peak in the range of 10-100 um, representing partial coalescence of emulsion
294
droplets compared to the fresh emulsion for all samples (Fig.4b) (Boode & Walstra, 1993). The
295
SPHPa thawed sample showed the highest volume in the second peak, followed by SMP and
296
CSPI thawed samples. The larger particles in the thawed sample of SPHPe was lowest among all
297
samples, indicating less fat globule coalescence (Fig.4b). d4,3 of SPHPa and SMP showed no
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significant difference in the fresh mix and the thawed ice cream mix (Fig 3). The particle size
299
(d4,3) of fat globules in SMP ice cream in this study was in accordance with the finding by Sung
300
& Goff (2010). All thawed samples had a significantly increased d4,3 compared to the fresh
301
samples with exception of SPHPe (Fig.3). SPHPe sample had the smallest increase in d4,3
302
amongst all samples, from 2.95 um to 4.69 um. The highest fat globules aggregation occurred in
303
samples made with SPHPa, where d4,3 increased from 1.99 um to 9.45 um. d4,3 of thawed ice
304
cream made with CSPI and NSPI were not significantly different from SMP, indicating similar
305
fat globules partial coalescence was formed during freezing compared to SMP. The degree of fat
306
destabilization was controlled by the properties of the fat crystal and the adsorbed protein at the
307
oil-water interface (Segall & Goff, 2002; Vanapalli, Palanuwech, & Coupland, 2002). Unsalted
308
butter used as the fat source consisted of high and low melting point fat triacylglycerol, existing
309
as a mixture of 2/3 crystalline and 1/3 liquid at 4°C (Adleman & Hartel, 2001), which ensured
310
the occurrence of partial coalescence with desired fat aggregated structure (Sung & Goff, 2010).
311
The mechanical properties of adsorbed protein film at oil/water interface and steric repulsion
312
between protein molecules prevented fat globules from coalescences in the aqueous phase (Bos
313
& Vliet, 2001). Most proteins used as the surfactant at oil/water interfaces decrease surface
314
tension about 15-20 mNm-1 but small surfactants can lower by 30-40 mNm-1 (Damodaran, 2005).
315
The displacement of adsorbed protein by emulsifier occurs because emulsifier has lower
316
interfacial tension at water/oil interface. As result, the weakened layer provides the condition for
317
the fat globules destabilization in dynamic freezing (Boode & Walstra, 1993; Gelin, Poyen,
318
Courthaudon, Le , & Lorient, 1994; Pelan, Watts, Campbell, & Lips, 1997). In this study, 0.1%
319
monoglycerides displaced soy protein and its hydrolysates from the fat globules surface during
320
mix aging except SPHPe. The high percentage of β-subunit in SPHPe kept the ice cream
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emulsion stable and cannot be displaced by monoglycerides. The quantities of displaced protein
322
and the profile change of protein at oil/water interface for all samples have been discussed in
323
Section 3.1 and further in 3.5.
324
3.5. Adsorbed protein fraction and composition
325
The extent of fat globule partial coalescence in ice cream mix has a direct relationship with the
326
adsorbed protein layer at oil/water interface (Bolliger et al., 2000). Measuring the change in
327
adsorbed milk protein amount at the oil/water surface in fresh ice cream mix and thawed ice
328
cream was used to predict the occurrence of partial coalescence during freezing (Barfod, Krog,
329
Larsen, & Buchheim, 1991; Gelin et al., 1994; Lai, O'Connor, & Eyres, 2006; Pelan et al., 1997).
330
Fig.5 shows the adsorbed protein fraction of the SMP, NSPI, CSPI, SPHPe and SPHPa at
331
oil/water in the fresh ice cream mixes and thawed ice cream mixes. Intact soy protein such as
332
NSPI and CSPI exhibited lower adsorbed amount at the interface compared to SPHPe and
333
SPHPa, which were attributed to the large molecule size and less molecule structure flexibility.
334
Limited enzymatic hydrolysis improved soy protein molecular flexibility and achieved better
335
emulsifying properties (Li et al., 2016). During ice cream aging, more protein desorption by
336
monoglycerides may lower steric stabilization of the fat globules to creates a thinner film, which
337
renders it prone to partial coalescence during ice cream freezing. After aging, the fraction of
338
absorbed protein on the fat globules decreased in all ice cream mix. The displaced fractions of
339
protein in ice cream mixes in this study were in the range of 8.0% to 22.6%. The largest fraction
340
of protein at the fat globules displaced during aging and freezing was SPHPa mix (22.6%),
341
which was comparable to that of SMP (21.8%). The lowest fraction displacement was SPHPe
342
mix (8.0%). Gelin et al. (1994) indicated milk protein can be displaced by monoglycerides from
343
oil/water interface during ice cream mix aging due to formation of favorable interface. For soy
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protein, all subunits and peptides can be displaced by monoglycerides except β–subunit due to its
345
strong adsorption ability in oil/water interface (Fig. 1b). Hence the protein composition absorbed
346
on fat globules indicated the emulsifier functionality at oil-water interface and the degree of
347
steric stabilization of fat globules. Pelan et al. (1997) found the emulsion stabilized by whey
348
proteins exhibited higher partial coalescence compared to skim milk powder because the
349
adsorbed layer formed by whey protein molecules was not as thick as casein micelles. For NSPI,
350
CSPI, SPHPa and SMP mixes, a large amount protein was displaced by monoglycerides, which
351
led to a lower steric stabilization of the droplet and rendered it to be more prone to partial
352
coalescence (Fig.3). SPHPa displayed an intermediate adsorbed protein load (Fig. 5), a high fat
353
particle size after freezing and thawing (Fig. 3) and consequently a low meltdown rate (Fig. 2),
354
all similar to SMP (Fig. 2). SPHPe with high percentage of β-subunit kept the emulsion stable
355
and only small portion of protein was displaced by monoglycerides (Fig.5), resulting in less
356
partial coalescence. (Fig. 3) and high meltdown rate (Fig. 2). In this case, since there was
357
insufficient partial coalescence of fat globules to stabilize the aqueous lamella between air cells
358
and prevent drainage, the serum inside the ice cream structure drained at a faster rate. Two
359
mechanisms were given to describe the mechanism of the displacement. Hoffmann & Reger
360
(2014) reported surfactants interacted with protein molecules at oil/water interface, resulting in
361
increased protein hydrophilic properties and desorbing from the interface. Another mechanism
362
was that emulsifiers were adsorbed into localized protein film defects, accumulating and
363
developing a domain, which forced weak adsorbed surface proteins towards aqueous phase
364
(Mackie et al., 2003). However, further study for the displacement mechanism of soy protein
365
molecules and monoglycerides at oil/water interface is advisable.
366
3.6. Confocal Laser scanning microscopy (CLSM)
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Confocal laser scanning microscopy (CLSM) was used to visually examine the structure of fat
368
globules in the melted ice cream mixes. The fat globules of thawed ice cream mixes made with
369
SMP, NSPI, CSPI and SPHPa interact with each other to form larger particles at different degree,
370
indicating partial coalescence of the milkfat globules compared to the fresh samples (Fig.6).
371
Clumps of fat globules were present in the serum phase, indicated by the intense red regions in
372
the micrographs. Partially coalesced fat globules covered the air interface incompletely and
373
stabilized air bubble in ice cream structure due to its hydrophobicity (Koxholt, Eisenmann, &
374
Hinrichs, 2001). Fig.6 demonstrates that there was a lot of fat agglomeration in the micrograph
375
of ice cream with SPHPa, similar to that shown by light scattering in Fig.3, which suggests a
376
potential alternative protein source in making ice cream. There are few fat globule aggregates
377
formed in ice cream made with SPHPe due to its stable emulsion.
378
4.0. Conclusion
379
This study presents new information on the role of soy protein isolate and its hydrolysates in ice
380
cream structure formation, including soy protein composition profile at the fat interface and ice
381
cream physico-chemical properties. The composition of soy protein isolate had a significant
382
impact on the degree of fat destabilization and melt-down rate of the ice cream. The relatively
383
high proportion of β-subunits in SPHPe prevented the occurrence of sufficient fat globules
384
partial coalescence and had the highest melting rate in all samples. The displacement of adsorbed
385
α', α, acidic and basic subunit in CSPI, NSPI and SPHPa on the fat globules by monoglycerides
386
promoted formation of fat partial coalescence and contributed to ice cream microstructure with
387
desirable melting properties. The meltdown and rheological properties of ice cream made with
388
SPHPa were comparable to SMP, which suggests a potential alternative to SMP for ice cream
389
manufacture.
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Conflict of interest
391 392
The authors state that there are no conflicts of interest regarding publication of this article.
393
Acknowledgement
394
This work was supported by the National Natural Science Foundation of China (Grant No.
395
31471583) and National First-class discipline program of Food Science and Technology (Grant
396
No. JUFSTR20180201).
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397
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Figure Captions
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Fig. 1. SDS-PAGE patterns of soy protein and its hydrolysates (a-ingredient, b- absorbed protein on the fat globules surface). BS: basic subunits; AS: acidic subunits; AB: their aggregates. α, α' and β are subunits from β-conglycinin.
536 537 538
Fig. 2. Meltdown rate of ice cream. Values are means ± standard deviation (the error between multiple measurements); values with same superscript letter are not significantly different (p > 0.05).
539 540 541
Fig. 3. The volume weighted mean particle diameter (d4,3) of ice cream with different soy protein isolate. Values are means ± standard deviation (the error between multiple measurements); values with same superscript letter are not significantly different P > 0.05.
542
Fig. 4. Droplet size distribution of (a) fresh ice cream mix and (b) thawed ice cream
543 544
Fig. 5. Absorbed protein fraction at the surface of fat globules. Values are means ± standard deviation (the error between multiple measurements)
545 546 547
Fig. 6. Microstructure of (a) fresh ice cream mix and (b) thawed ice cream with SMP, NSPI, CSPI, SPHPe and SPHPa. Bar r = 25 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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ACCEPTED MANUSCRIPT Table 1 Formulations of ice cream mixes containing different protein Ingredients
SMP
NSPI
CSPI
SPHPe
SPHPa
(%)
(%)
(%)
(%)
(%)
10.00
10.00
10.00
10.00
10.00
Fat
10.00
10.00
10.00
10.00
10.00
CSS
4.00
4.00
4.00
4.00
4.00
SMP
10.00
0
0
0
0
NSPI
0
4.40
0
CSPI
0
0
4.30
SPHPe
0
0
0
SPHPa
0
0
0
Lactose
0
5.60
5.70
Stabilizers
0.15
0.15
0.15
Monoglycerides
0.15
0.15
0.15
IW
65.70
65.70
65.70
100
100
100
a
0
0
0
5.15
0
0
4.50
4.85
5.50
0.15
0.15
0.15
0.15
65.70
65.70
100
100
SC
0
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Total
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Sugar
Abbreviations: NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate; SPHPe, Soy Proteins
Isolate hydrolyzed by Pepsin; SPHPa: Soy protein Isolate hydrolyzed by Papain; CSS, Corn Syrup Solid; IW:
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Ingredient Water.
ACCEPTED MANUSCRIPT Table 2 Properties of soy protein isolate and its hydrolysates EAI (m2/g)
DH
ESI (%)
SMP
-
33.96 ± 0.32c
85.06 ± 0.51b
NSPI
0.0% ± 0.1a
36.69 ± 1.04b
89.63 ± 1.20ab
CSPI
2.4% ± 0.2b
32.65 ± 0.16c
86.68 ± 4.65b
SPHPe
3.6% ± 0.4c
42.21 ± 0.99a
94.72 ± 0.88a
SPHPa
15.6% ± 0.7d
40.43 ± 0.72a
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Ingredients
63.20 ± 0.81c
*DH: Degree of hydrolysis; EAI: Emulsifying activity index; ESI: emulsion stability index. Values with the same superscript letters within each column are not significantly different (P>0.05). DH is not tested for SMP.
SC
Abbreviations: SMP, Skim milk powder; NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate;
AC C
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SPHPe, Soy Proteins Isolate hydrolyzed by Pepsin; SPHPa, Soy Protein Isolate hydrolyzed by Papain.
ACCEPTED MANUSCRIPT Table 3 The densitometric analysis: relative compositions (%) of soy proteins and hydrolysates in both the starting ingredient materials and adsorbed to the fat globules as isolated from melted ice cream α'-
Subunit
α
β
AB
AS
BS
11.5
6.1
Ingredient
5.5
6.3
11.2
24.9
Adsorbed protein
4.5
5.4
10.8
27.0
Ingredient
9.5
13.1
4.1
0.0
Adsorbed Protein
6.5
6.6
3.9
0.0
Ingredient
25.4
8.7
14.3
Adsorbed Protein
10.1
7.0
13.9
Ingredient
0.0
0.0
0.0
Adsorbed protein
0.0
0.0
0.0
CSPI
5.3
21.5
0.0
5.8
0.0
0.0
4.1
17.9
0.0
0.0
4.2
0.0
0.0
0.0
0.0
0.0
0.0
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SPHPa
7.8
SC
SPHPe
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NSPI
*Abbreviations: NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate; SPHPe, Soy Protein Isolate hydrolyzed by Pepsin; SPHPa, Soy Protein Isolate hydrolyzed by Papain. BS: basic subunits; AS: acidic
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subunits; AB: their aggregates. α, α' and β are subunits from β-conglycinin.
ACCEPTED MANUSCRIPT Table 4 Rheological properties of ice cream mixes Herschel-Bulkley Parameter K (Pa Sn)
Apparent Viscosity (Pa s)
n
τ0
10 s-1
100 s-1
SMP
0.64±0.24b
0.58±0.03ab
0.19±0.05a
0.26
0.09
NSPI
1.23±0.43b
0.55±0.03a
0.35±0.09a
0.47
0.16
CSPI
b
0.74±0.22
0.49±0.06
a
0.24
0.07
SPHPe
2.12±0.96a
0.55±0.05ab
SPHPa
b
0.46±0.03
c
0.15±0.09
0.72±0.39a a
0.32±0.01
RI PT
0.86±0.03
bc
0.82
0.27
0.28
0.07
* K = consistency index; n = flow behavior index; τ0 = Yield Stress. Values with the same superscript letters within each column are not significantly different (P>0.05). Abbreviations: SMP, Skim milk powder; NSPI, Native Soy Protein Isolate; CSPI, Commercial Soy Protein Isolate; SPHPe, Soy Proteins Isolate hydrolyzed by Pepsin; SPHPa,
AC C
EP
TE D
M AN U
SC
Soy protein Isolate hydrolyzed by Papain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
SMP
NSPI
NSPI
CSPI
CSPI
M AN U
SC
SMP
RI PT
ACCEPTED MANUSCRIPT
SPHPe
TE D
SPHPe
SPHPa
AC C
EP
SPHPa
(a)
(b)
ACCEPTED MANUSCRIPT Highlight 1. Selective hydrolysis led to hydrolysates with the different polypeptide profile.
2. The interaction between SPI subunits and emulsifier decided meltdown properties.
RI PT
3. SPHPe ice cream show insufficient fat partial-coalescence and rapid melting rate.
AC C
EP
TE D
M AN U
SC
4. Ice cream with SPHPa exhibited the similar melting property as milk protein.